Genetic disorders are caused by diverse types of mutations. However, many of these mutations, including nonsense mutations, frameshift mutations, and mutations that cause alternative splicing events, result in premature termination codons (“PTC”s). For example, approximately 15% of the mutations in the gene responsible for Duchenne muscular dystophy (“DMD”), a genetic disease that affects one in 3500 males and is characterized by severe, rapid muscle degeneration, are single base changes or multi-exon deletion within exons 44-55 of the DMD gene which result in PTCs (Aartsma-Rus et al., “Entries in the Leiden Duchenne Muscular Dystrophy Mutation Database: An Overview of Mutation Types and Paradoxical Cases that Confirm the Reading-Frame Rule,” Muscle Nerve 34:135-144 (2006) and Dent et al., “Improved Molecular Diagnosis of Dystrophinopathies in an Unselected Clinical Cohort,” Am. J. Med. Genet. A. 134:295-298 (2005)). In Sardinia, almost all cases of thalassemia major, a devastating disease characterized by severe anemia, splenomegaly, and iron overload, is caused by a PTC mutation in codon 39 of the β globin gene (Kan et al., “Polymorphism of DNA Sequence in the Beta-Globin Gene Region. Application to Prenatal Diagnosis of Beta 0 Thalassemia in Sardinia,” N. Engl. J. Med. 302:185-188 (1980) and Maquat et al., “Unstable Beta-Globin mRNA in mRNA-Deficient Beta O Thalassemia,” Cell 27:543-553 (1981)). In cystic fibrosis (“CF”), a multi-organ disease most marked by pulmonary dysfunction, 10% of the mutations in cystic fibrosis transmembrane conductance regulator (“CFTR”) gene worldwide (Bobadilla et al., “Cystic Fibrosis: A Worldwide Analysis of CFTR Mutations—Correlation with Incidence Data and Application to Screening,” Hum. Mutat. 19:575-606 (2002)), and most of the mutations in Israel (Kerem et al., “Cystic Fibrosis in Jews: Frequency and Mutation Distribution,” Genet. Test. 1:35-39 (1997)) are nonsense mutations. And 70% of the mutations in enzyme α-L-iduronidase, responsible for Hurler's disease, are PTC mutations (Bunge et al., “Mucopolysaccharidosis Type I: Identification of 8 Novel Mutations and Determination of the Frequency of the Two Common Alpha-L-Iduronidase Mutations (W402X and Q70X) Among European Patients,” Hum. Mol. Genet. 3:861-866 (1994)). Together, it has been estimated that up to 30% of all mutations resulting in human genetic disorders result in PTCs (Frischmeyer et al., “Nonsense-Mediated mRNA Decay in Health and Disease,” Hum. Mol. Genet. 8:1893-1900 (1999)). Although screening has reduced the prevalence of these diseases (Kan et al., “Polymorphism of DNA Sequence in the Beta-Globin Gene Region. Application to Prenatal Diagnosis of Beta 0 Thalassemia in Sardinia,” N. Engl. J. Med. 302:185-188 (1980)) and supportive treatments can improve complications of these disorders, in most cases no direct disease-modifying treatments are available.
Many transcripts carrying a PTC are targeted for rapid degradation before they can be translated into protein through a multistep process termed nonsense mediated RNA decay (“NMD”). During the processing of mammalian pre mRNA, introns are excised and marked by a multi-protein complex termed the exon junction complex (“EJC”) (Lykke-Andersen et al., “Communication of the Position of Exon-Exon Junctions to the mRNA Surveillance Machinery by the Protein RNPS1,” Science 293:1836-1839 (2001); Lykke-Andersen et al., “Human Upf Proteins Target an mRNA for Nonsense-Mediated Decay When Bound Downstream of a Termination Codon,” Cell 103:1121-1131 (2000); and Modrek et al., “A Genomic View of Alternative Splicing,” Nat. Genet. 30:13-19 (2002)). When the translation complex pauses at a PTC that is upstream of an EJC, eukaryotic release factors physically bind to and recruit the RNA helicase Upf1, a vital component of the NMD mechanism (Czaplinski et al., “The Surveillance Complex Interacts with the Translation Release Factors to Enhance Termination and Degrade Aberrant mRNAs,” Genes Dev. 12:1665-1677 (1998); Gehring et al., “Y14 and Hupf3b Form an NMD-Activating Complex,” Mol. Cell 11:939-949 (2003); Lykke-Andersen et al., “Human Upf Proteins Target an mRNA for Nonsense-Mediated Decay When Bound Downstream of a Termination Codon,” Cell 103:1121-1131 (2000); and Serin et al., “Identification and Characterization of Human Orthologues to Saccharomyces cerevisiae Upf2 Protein and Upf3 Protein (Caenorhabditis elegans SMG-4),” Mol. Cell Biol. 21:209-223 (2001)). Subsequently, the Upf1 containing complex at the PTC bridges with components in the EJC which promotes the phosphorylation of Upf1. Phosphorylated Upf1 then recruits SMG7, with the subsequent de-phosphorylation of Upf1 by SMG7 (Fukumura et al., “Tumor Microvasculature and Microenvironment: Targets for Anti-Angiogenesis and Normalization,” Microvasc. Res. 74:72-84 (2007)). The de-phosphorylation of Upf1 by SMG7 is a necessary step in the NMD pathway, prior to the transcript's degradation by exonucleases (Kashima et al., “Binding of a Novel SMG-1-Upf1-Erf1-Erf3 Complex (SURF) to the Exon Junction Complex Triggers Upf1 Phosphorylation and Nonsense-Mediated mRNA Decay,” Genes Dev. 20:355-367 (2006); Ohnishi et al., “Phosphorylation of hUPF1 Induces Formation of mRNA Surveillance Complexes Containing hSMG-5 and hSMG-7,” Mol. Cell 12:1187-1200 (2003); and Yamashita et al., “Human SMG-1, a Novel Phosphatidylinositol 3-Kinase-Related Protein Kinase, Associates with Components of the mRNA Surveillance Complex and is Involved in the Regulation of Nonsense-Mediated mRNA Decay,” Genes Dev. 15:2215-2228 (2001)). Despite insights into the molecular mechanism of NMD, no effective strategy for the pharmacological inhibition of NMD has been developed.
Even a small amount of full length functional protein expressed from the gene containing a PTC may be sufficient to improve the clinical symptoms of a variety of genetic conditions. For example, hemophilia A (which can be marked by PTC mutations) can be effectively treated if the amount of functional factor VIII protein is increased to even 5% of normal (White et al., “Definitions in Hemophilia. Recommendation of the Scientific Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis,” Thromb. Haemost. 85:560 (2001)) and 5% of normal CFTR mRNA is sufficient to ameliorate the pulmonary effects in CF (Ramalho et al., “Five Percent of Normal Cystic Fibrosis Transmembrane Conductance Regulator mRNA Ameliorates the Severity of Pulmonary Disease in Cystic Fibrosis,” Am. J. Respir. Cell Mol. Biol. 27:619-627 (2002)). Unfortunately, the difficulties in effectively and safely achieving these goals with the attractive strategy of gene therapy are well documented. Thus several other strategies have been pursued.
One such approach to treat genetic disorders with PTC mutations is to pharmacologically promote ribosomes to read-through a PTC and produce a full-length protein (Burke et al., “Suppression of a Nonsense Mutation in Mammalian Cells In vivo by the Aminoglycoside Antibiotics G-418 and Paromomycin,” Nuc. Acids Res. 13:6265-6272 (1985)). For example, treating cells engineered to express a β globin PTC mutated gene with one such agent, the antibiotic gentamicin, leads to real, although small, increases in β□ globin expression (Salvatori et al., “Production of Beta-Globin and Adult Hemoglobin Following G418 Treatment of Erythroid Precursor Cells from Homozygous Beta(0)39 Thalassemia Patients,” Am. J. Hematol. 84:720-728 (2009)). Several studies also have shown that gentamicin treatment of cells with PTC mutations of the CFTR can increase cellular chloride transport (Bedwell et al., “Suppression of a CFTR Premature Stop Mutation in a Bronchial Epithelial Cell Line,” Nat. Med. 3:1280-1284 (1997) and Howard et al., “Aminoglycoside Antibiotics Restore CFTR Function by Overcoming Premature Stop Mutations,” Nat. Med. 2:467-469 (1996)). In addition, CF patients with PTC mutations treated with intranasal gentamicin increase CFTR protein expression and improve the potential difference of nasal epithelial cells, while no such response was seen in CF patients with non-PTC mutations in the CFTR (Wilschanski et al., “Gentamicin-Induced Correction of CFTR Function in Patients with Cystic Fibrosis and CFTR Stop Mutations,” N. Engl. J. Med. 349:1433-1441 (2003)).
Because gentamicin is relatively toxic, is inconvenient to administer, and is weak at bypassing PTC mutations (Welch et al., “PTC124 Targets Genetic Disorders Caused by Nonsense Mutations,” Nature 447:87-91 (2007)), other agents have been developed. A recent high throughput screen identified a small molecule, Ataluren (PTC124/Ataluren) which has similar properties but appears to be more potent and less toxic (Welch et al., “PTC124 Targets Genetic Disorders Caused by Nonsense Mutations,” Nat. 447:87-91 (2007)) (although the initial screening strategy may have been flawed (Auld et al., “Mechanism of PTC124 Activity in Cell-Based Luciferase Assays of Nonsense Codon Suppression,” Proc. Natl. Acad. Sci. U.S.A. 106:3585-3590 (2009)). While this drug is being tested in several diseases, including CF, the preclinical use of this drug is most mature in muscular dystrophy. Specifically, Ataluren has been tested in dystrophin knockout mice which transgenetically produce a dystrophin cDNA with a PTC (Welch et al., “PTC124 Targets Genetic Disorders Caused by Nonsense Mutations,” Nat. 447:87-91 (2007)). Treatment of these mice with Ataluren led to increased expression of full-length dystrophin and an improvement in muscle strength. However, in this model the replacement dystrophin cDNA does not contain introns, and is thus not a NMD target, in contrast to naturally occurring mutations in the dystrophin gene in patients. The bypass of a PTC by Ataluren does not inhibit NMD (Dietz, H. C “New Theraputic Approaches to Mendelian Disorders,” N. Engl. J. Med. 363:852-863 (2010)), and thus in patients the mutated mRNA is still subjected to NMD. This may be one reason why, although phase II trials in cystic fibrosis and muscular dystrophy have shown promise (e.g., reduction in epithelial electrophysiological abnormalities in CF patients (Kerem et al., “Effectiveness of PTC124 Treatment of Cystic Fibrosis Caused by Nonsense Mutations: A Prospective Phase II Trial,” Lancet. 372:719-727 (2008)) and improved walk-times in DMD patients (http://ptct.client.shareholder.com/releasedetail.cfm?ReleaseID=518941)), clinical improvements have largely been modest. Thus, alternative or additional treatments may be necessary to make PTC by-pass drugs like Ataluren more effective.
Additional strategies have been employed to compensate for other mutations, which also result in PTCs in various disorders. For example, exon deletions seen in up to 70% of all DMD patients are generally in the rod domain of dystrophin and generate PTCs. Because in-frame deletions of the rod domain result in the clinically milder disease Becker muscular dystrophy, restoration of the reading frame in these DMD patients has been pursued (reviewed in Nelson et al., “Emerging Genetic Therapies to Treat Duchenne Muscular Dystrophy,” Curr. Opin. Neurol. 22:532-538 (2009)). Strategies such as the use of antisense olignonucleotides to promote exon skipping are being tested in stage I/II studies (reviewed in Nelson et al., “Emerging Genetic Therapies to Treat Duchenne Muscular Dystrophy,” Curr. Opin. Neurol. 22:532-538 (2009)).
Bypassing PTCs via drugs that either promote PTC read-through, or strategies that promote exon skipping, are likely to be more effective with higher cellular concentrations of PTC mutated mRNA. Indeed, in vitro experiments have demonstrated that the expression of PTC mutated CFTR, as well as CFTR mediated chloride transport, are improved in gentamicin treated cells when Upf1 or Upf2 are also depleted (Linde et al., “Nonsense-Mediated mRNA Decay Affects Nonsense Transcript Levels and Governs Response of Cystic Fibrosis Patients to Gentamicin,” J. Clin. Invest. 117:683-692 (2007)). Thus, it is hypothesized that the combination of a drug that can bypass a PTC and/or promote exon skipping in combination with a drug that inhibits NMD will reflect a synergistic combination and serve as an effective platform to treat genetic disease. Previously, no pharmacological approach to inhibit NMD has been demonstrated (Welch et al., “PTC124 Targets Genetic Disorders Caused by Nonsense Mutations,” Nat. 447:87-91 (2007)).
The present invention is directed to overcoming these and other deficiencies in the art.